Composition for feeding prey organisms in aquaculture

ABSTRACT

A composition is for feeding aquacultural prey organisms such as  Artemia  and rotifiers, comprising a lipid component comprising at least 25 wt % of phospholipids and providing a DHA content of at least 30 wt %. The lipid component is preferably derived from marine organisms such as fishmeal, phytoplankton or zoo plankton biomass. The composition is used for providing prey organisms having a high content of highly-unsaturated fatty acids (HUFAs) suitable for aquaculture of fish including halibut, turbot, bass and crustaceans and molluses.

FIELD OF THE INVENTION

The present invention is within the field of aquaculture, in particularthere is provided a lipid composition for feeding prey organisms whichare used as feed for fish larvae and larvae of crustaceans and bivalves.More specifically, the invention provides marine lipid compositionswhich are highly enriched in their content of highly unsaturated fattyacids (HUFAs), in particular docosahexaenoic acid (DHA) and having ahigh content of DHA-rich phospholipids.

TECHNICAL BACKGROUND AND PRIOR ART

The consumption of seafood species for which there is a high consumerdemand such as salmon, trout, halibut and eel is increasing and due tothis high demand and limited natural stocks, much effort is spent ondeveloping cost effective aquacultural methods of farming such species.A particularly serious problem is to secure a high survival rate of thehatched larvae of the species being cultivated.

Expansion of the aquaculture industry requires that several problems beaddressed, one of the most significant being the difficulty of supplyinglive prey organisms which provide a nutritionally adequate feed for thelarvae. Larval fish in the wild consume a mixed population ofphytoplankton prey organisms that provide a balanced nutrition. However,collecting phytoplankton in sufficient quantities to meet the demand inaquaculture is not feasible. As an alternative, selected species of preyorganisms, in particular rotifers and Artemia species, are presentlycultivated and used as feed.

Generally however, such artificially cultivated prey organisms, althoughthey provide adequate amounts of protein and energy, have a lipidcomposition which is not adequate to cover the requirement for certainHUFAs, in particular DHA and EPA which are essential for the optimumsurvival, growth and development of larvae. Specifically, it has beenshown that a high content of DHA is required and that the ratio betweenDHA and EPA in the prey organisms should be at least 1:1 and preferablyat least 2:1. To provide prey organisms having such a composition inrespect of HUFAs it is necessary to cultivate the organisms in thepresence of enrichment compositions having a high content of DHA,preferably at least 20 wt % and a ratio of DHA to EPA exceeding theratio aimed at in the prey organisms, such as at least 3:1 andpreferably higher.

Currently, this problem is being addressed by cultivating the preyorganisms in the presence of enrichment compositions permitting theorganisms to be enriched in respect of these essential fatty acids.However, presently available commercial compositions for that purposesuch as emulsion products sold under the tradename Selco™, do not meetthe above requirements in that the DHA content is relatively low and/orthe DHA:EPA ratio is not high enough. Using Artemia enriched with thesecommercial compositions survival rates of fish larvae in the range of 12to 15% have been reported (McEvoy et al. Aquaculture 163 (1998) 237–250;Navarro et al. J. Fish Biol. 43 (1993) 503–515). In this context,survival rates are defined as survival percentage from the first feedingthrough metamorphosis. For cost-effective aquaculture production alarval survival rate of 50% and preferably higher should be obtained.

Other commercially available compositions for prey organism enrichmentare products sold under the tradename Algamac™ containing up to 14 wt %of DHA, and tuna orbital oil (TOO) that contains up to 30 wt % of DHA.

WO 99/37166 discloses a method for the enrichment of live prey organismswith nutrients essential for fish larvae based on the use of dry fattyacid soap powders of HUFAs obtained from the waste stream of marinealgae oil extraction. The raw material for providing these powders has acontent of phospholipids and about 28 wt % of free fatty acids and itcontains about 23 wt % of DHA but apparently no other n-3 fatty acids.Artemia enrichment levels of about 2.7% DHA of dry weight are disclosed.

Another material intended for use in aquaculture is described in WO99/06585. Examples disclose a DHA content of 24 wt %, but thephospholipid content is not disclosed. The material however, contains ahigh proportion of free fatty acids (about 32–37 wt %) and a highcontent of non-lipid material (about 39–44 wt %), which may reduce thelipid uptake efficiency of prey animals. A high content of free fattyacids is generally considered harmful for fish larvae and juveniles.

Neither of the two last-mentioned materials is fish-based and they lackmany HUFAs found in fish, such as EPA and other n-3 fatty acids.

In a recent review by Sargent et al. (Aquaculture 179 (1999) 217–229) itis emphasized that in addition to the requirement in respect of HUFAs,fish larvae have a dietary requirement for phospholipids and it isstressed that the ideal diet for fish larvae is a diet having acomposition similar to the yolk of the eggs. According to these authorsfish egg yolk contains about 10 wt % (on a dry matter basis)phospholipids which contain about 17 wt % of DHA an about 9 wt % of EPA.These authors conclude in their review that a problem remains withrespect to how to construct such a diet on a commercial scale fromcurrently available materials.

To our knowledge, it has not been possible, with the use of theabove-mentioned commercial feed compositions, or other prior artexperimental compositions, to obtain DHA enrichment levels in Artemiathat approach an ideal, egg yolk-similar diet.

It has now been found that it is possible to provide a lipid compositionfor enriching aquacultural prey organisms based on the use of DHA-richphospholipids isolated from abundantly available marine organismmaterials such as fish meal. By using this starting material it hasbecome possible to provide enrichment compositions on a commercialscale, which make it possible to provide prey organisms having, inrespect of HUFAs and phospholipids, a composition which is very close tothat of fish egg yolk and which are therefore highly appropriate tosecure optimum survival, growth, pigmentation and morphogenesis of fishlarvae such as halibut larvae.

SUMMARY OF THE INVENTION

Accordingly, in a first aspect the present invention pertains to acomposition for feeding prey organisms for use in aquaculture,comprising a lipid component comprising at least 25 wt % ofphospholipids, the composition providing a DHA content of at least 30 wt%. In useful embodiments the source of the phospholipid-rich lipidcomponent is abundantly available marine organism materials such asfishmeal. This composition according the invention is particularlyuseful for providing Artemia species and rotifers having an appropriatelipid composition for fish larvae.

In a further aspect the invention provides an emulsion comprising as thelipid phase the above composition.

In a still further aspect the invention relates to method ofmanufacturing a composition as defined above, comprising the steps ofseparating from a marine organism material a crude lipid componentcomprising triglycerides and phospholipids, followed by a phospholipidenrichment step comprising adding, at a temperature where triglyceridesdo not precipitate, a solvent to the crude lipid component and coolingthe mixture to precipitate triglycerides to obtain a lipid fractioncomprising at least 25 wt % of phospholipids.

There is also provided a marine lipid composition comprising at least 50wt % of phospholipids.

DETAILED DESCRIPTION OF INVENTION

The composition according to the invention, for feeding prey organismscomprises as its main component a phospholipid-rich component.

In the present context, the expression ‘prey organisms’ refers to anymarine organism that can be used as live feed for larvae of marinespecies that are produced in aquacultural facilities. A general reviewof such prey organisms can be found in Lavens & Sorgeloos (eds.) “Manualon the production and use of live food for aquaculture” published by FAO(1995) which is hereby incorporated by reference. Accordingly, the mostcommonly used prey organisms include several classes and genera ofmicroalgae, rotifers, Artemia, zooplankton including copepods,cladocerans, nematodes, and trochophora larvae.

As used herein, the term ‘aquaculture’ is to be understood in itsbroadest sense and it includes any production of any aquatic speciesproduced under aquacultural conditions, such as fish species, includingas examples salmon, trout, carp, bass, bream, turbot, sole, milkfish,grey mullet, grouper, bream, halibut; crustaceans such as shrimp,lobster, crayfish and crabs; molluscs such as bivalves.

A common feature of these aquatic species is that the life cycleincludes one or more larval stages which may have very specificnutritional requirements and accordingly the provision of live preyorganisms meeting this requirement is an essential factor for successfulaquacultural production. As mentioned above, one such specificrequirement is a high content of the essential fatty acid DHA, the term‘essential’ implying that the prey organisms are not capable of de novosynthesis of such compounds. The composition of the present inventionhas a DHA content of at least 30 wt %, such as in the range if about 30to 35 wt %, or the range of about 35–40 wt % preferably at least 40 wt%, such as at least 50 wt % including at least 60 wt %.

In order to provide a composition having this high content of DHA, thelipid components of the composition must be selected so as to providethe desired amount of DHA. In useful embodiments, the phospholipid-richcomponent provides the required amount of DHA. However, in otherembodiments the DHA content of the phospholipid component may not besufficient. In such cases, at least one further lipid component thatprovides an adequate amount of DHA must be incorporated. Typically, sucha further lipid component contains at least 20 wt % of DHA, preferablyat least 30 wt % of DHA, more preferably at least 40 wt % of DHA, forexample at least 50 wt % of DHA. In preferred embodiments, the furtherlipid component includes at least 60 wt % of DHA, such as at least 70 wt% DHA, including at least 90 wt % DHA.

If required, the further DHA-rich lipid component is incorporated in thecomposition of the invention in an amount that at least results in atotal DHA content of the composition which is at least 30 wt %.Depending on the DHA content of the phospholipid-rich component, theamount of the further lipid component required may be in the range of5–99 wt %. In certain embodiments of the invention the amount of thefurther lipid component is in the range of 50–95 wt %, such as a rangeof about 60–80 wt %, such as e.g. 70–75 wt %.

The source of the further lipid component may be any naturally occurringlipids (preferably comprising glycerides, such as triglycerides ordiglycerides) containing at least 20 wt % of DHA and any such lipidssynthesized chemically or enzymatically. Examples of naturally occurringDHA-rich lipid are tuna orbital oil (TOO) and lipids isolated frommicrobial cells having a high content of DHA, such as algae includingChlorella and Crypthecodinium species, certain yeast species such asSaccharomyces, Morteriella, Schizochytrium and Thraustochytrium. As analternative to using naturally occurring DHA-rich lipids, such lipidscan be synthesized chemically or enzymatically. In a useful embodimentof the invention such a lipid is provided as a glyceride by contactingDHA as a free fatty acid and glycerol in the presence of chemicalcatalysts or an enzyme capable of forming glycerides from the reactants,such as a lipase including a lipase isolated from Candida antarctica.

It has been found particularly useful to use fish-based materials aslipid sources for the composition according to the invention (e.g., assources for the phospholipid containing component, or of the furtherDHA-rich component, or both). In addition to DHA, such materials alsoprovide other valuable HUFAs (particularly n-3 HUFAs) that arecharacteristic of fish (such as 20:5 (EPA), 18:3, 18:4, and 20:4 fattyacids), and which are found in the natural diet of fish larvae and infish egg yolk, and are considered beneficial for successful enrichmentof prey organsims for rearing of fish. In an advantageous embodiment theenrichment composition has a total content of HUFAs of at least 30 wt %,for example at least 40 wt %, preferably at least 50 wt %, such as atleast 60 wt %. In such compositions, EPA is preferably in the range of2–15 wt %, such as in the range of 5–10 wt %, and other n-3 HUFAs thanDHA and EPA are preferably in the range of 2–25 wt %, preferably in therange of about 5–15 wt %, such as e.g. about 10 wt %. In furtheradvantageous embodiments the total content of n-3 HUFAs is at least 50wt %, such at least 60 wt %.

As mentioned above, the composition of the invention comprises as amajor component a phospholipid-rich component comprising at least 25 wt% phospholipids. In the present context the term ‘phospholipids’ is usedto describe a class of lipids containing phosphoric acid as a mono ordiester. Thus, phospholipids include phosphatidyl choline (PC),phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), phosphatidylglycerol (PG), phosphatidyl serine (PS), and phosphatidic acid (PA). Theterm ‘lecithin’ is also commonly used for mixtures of the abovephospholipids. The present inventors have found that the distribution ofthe members of phospholipid class may have significant impact on theability of the composition of the invention to secure high survival andgood pigmentation rates of fish larvae. In particular, it has been foundthat the composition of the phospholipids in herring meal is such that aparticularly high rate of correct pigmentation of fish larvae isachieved.

In accordance with the invention any phospholipid-rich component can beused. However, presently preferred phospholipids are phospholipidsisolated from marine organism materials, including fresh materials anddried materials. Fresh materials include for example viscera from fishand other marine animals, flesh of fish, fish eggs, squids, molluscs anda planktonic biomass. Dried materials include, in particular, fishmealssuch as meals of herring, capelin, mackerel, menhaden, sardine, anchovy,horse mackerel, and blue whiting and meals of planktonic organisms. Suchmarine source materials from fish sources, provide as mentioned earlier,not only a high content of DHA, but also EPA and other n-3 PUFAs,characteristic of fish, and are to an extent similar to the natural dietof fish larvae.

It is well known that the quality of commercial fishmeals can varyconsiderably. In particular, fish meals of poor quality have undergonemore or less advanced deterioration of the lipids including oxidationand hydrolysis. For the purposes of the present invention it isparticularly preferred to use commercial fishmeals of a high quality asa source of the phospholipid-containing component. High quality fishmeal is defined in this context is with the following parametersrelating to quality and freshness: ‘total volatile nitrogen’ (TVN)should be less than 50 mg/100 g measured as described in Antonacopoulus,N. Handbuch der Lebensmittelcheme, vol. III/2, Springer Veriag, Berlin(1968); and mink digestability of protein should be at least 90%.

In one preferred embodiment, the phospholipid-containing component has acontent of phospholipids of at least 30 wt %, preferably at least 40 wt%, such as at least 50 wt %, for example at least 60 wt %, including atleast 70 wt % of phospholipids, or even higher. Provided aphospholipid-containing component is selected that has a sufficientlyhigh DHA content, the composition of the invention may comprise thiscomponent as the sole lipid-containing component. However, as mentionedabove, it may be required to supplement the phospholipid-containingcomponent with a further lipid component that has a high content of DHA.In that case, the proportion of the phospholipid-containing compositionis in the range of 1–99 wt %, such as in the range of 2–75 wt %including the range of 5–50 wt %, such as in the range of 5–25 wt %,including about 5 wt %, about 10 wt %, and about 15 wt %, or in therange of about 25–50 wt %.

As mentioned above, it has been shown that not only a high content ofDHA is required in the feed for fish larvae, but the ratio between DHAand EPA in the prey organisms is of significance for the survival anddevelopment of the larvae of the cultured species. It is generallyrecognized that the DHA:EPA ratio in the prey organisms should be atleast 1:1 and preferably at least 2:1. However, it has been found thatin order to achieve this desired ratio in the prey organisms, asignificantly higher ratio may be required in the enrichment compositionfor the prey organism. Accordingly, the enrichment composition of theinvention has a DHA:EPA wt. ratio in the range of 1:1 to 10:1, such as2:1 to 8:1, including the range of 4:1 to 6:1.

As stated by Sargent et al., supra, other HUFAs may be of significance,such as for example arachidonic acid. Accordingly, an enrichmentcomposition according to the invention including a sufficient amount ofarachidonic acid is contemplated. If required, the content ofarachidonic acid in the composition can be in the range of 1–20 wt % ofthe total fatty acids, such as for example 2–10 wt %.

It is generally known in the state of the art, that a high content offree fatty acids may be harmful for fish juveniles. Preferredembodiments of the composition of the present invention have a lowcontent of free fatty acids, such as a total content of less than about10 wt %, including less than about 5 wt %, more preferably less thanabout 3 wt %, such as less than about 1 wt %.

In useful embodiments the enrichment composition of the inventioncomprises additional components including emulsifiers, immunostimulantssuch as for example glucans or alkoxyglycerols, vitamins, antioxidants,mineral, and arachidonic acid. Suitable emulsifiers include ChremophoreA25™ available from BASF, VOLPO™ from Croda, soy lecithin,phospholipids, glycerides and fatty acids including soaps.

Vitamins which may be incorporated in the composition include anywater-soluble vitamin such as vitamin B and vitamin C, and any waterinsoluble vitamin A, vitamin D, and vitamin E. It has been found that ahigh content of vitamin C may have a significant impact on theperformance of the enrichment composition with respect to the survivaland development of the larval predators (Merchie, G.; Lavens, P.;Sorgeloos, P. Effects of dietary vitamin C on fish and crustaceanlarvae, Proceedings Larvi'95, Ghent (1995); Merchie, G. Nutritionaleffect of vitamin C on the growth and physiological condition of thelarvae of aquaculture organisms, Thesis, 1995, University in Ghent;Merchie, G., et al. Aquaculture, 134 (1995) 325–337.) Accordingly, inuseful embodiments the content of vitamin C may be in the range of 1–15wt %, such as for example 5–10 wt %. Useful antioxidants include TBHQ,ethoxyquin, BHT, BHA, vitamin E, and vitamin C.

It will be appreciated by the skilled person that the enrichmentcomposition of the invention can be used as a convenient carrier forpharmaceutically active substances such as for example antimicrobialagents and immunologically active substances including vaccines againstbacterial or viral infections, and any combination hereof.

The composition according to invention can be provided as a liquid,pourable emulsion, or in the form of a paste, or in a dry form, forexample as a granulate, a powder, or as flakes. When the composition isprovided as an emulsion, preferably a lipid-in-water emulsion, it ispreferred that it is in a relatively concentrated form. Such aconcentrated emulsion form can also be referred to as a pre-emulsion asit can be diluted in one or more steps in an aqueous medium to providethe final enrichment medium for the prey organisms. In preferredembodiments, such a preemulsion comprises as the lipid phase at least 50wt % of the enrichment composition according to the invention.

As it appears from the above, the enrichment composition can be used forenriching aquacultural prey organisms with respect to essential HUFAsand phospholipids.

In a further aspect, the present invention provides a method ofmanufacturing an enrichment composition as described above. The methodcomprises in a first step the separation of a crude lipid component froma marine organism material as defined above. The separation can beaccomplished by any conventional method for separating lipids fromorganic material, such as an extraction using any suitable solvent,including organic solvents such as alcohols including ethanol andpropanols; hydrocarbon solvents e.g. alkanes such as a pentane, hexane,or mixture of alkanes or cyclic or aromatic hydrocarbon solvents;ethers;esters, or a mixture of these or other solvents found to besuitable. Use of supercritical extraction is also contemplated. Othermeans of separating phospholipids from the source materials may includechromatography, centrifugation, compression, thermal treatment or anycombination thereof. In a second step, the phospholipid fraction of thecrude lipid component is enriched by subjecting the lipid component to atreatment resulting in an at least partial precipitation orsolidification of the triglyceride fraction, which can subsequently beremoved. The treatment advantageously comprises the addition of asuitable solvent, such as one of those above-mentioned to the crudelipid component, at a temperature where the triglycerides do notprecipitate followed by cooling the lipid/solvent mixture to atemperature where a significant portion of the triglyceridesprecipitates. The precipitate is then removed and the solvent phase isevaporated to obtain a phospholipid-rich component. This treatment canbe repeated one or more times and the resulting aliquots ofphospholipid-enriched components can be combined. Suitable raw materialsfor providing the phospholipid-rich component include those marineorganism materials as described above.

As mentioned above, it may be required to supplement the phospholipidcomponent with a further lipid component having a high DHA content.Accordingly, the method of the invention includes the embodiment wherethe phospholipid component is combined with such a DHA-rich component asit is described above.

In a further aspect, the invention provides a novel marine lipidcomposition comprising at least 50 wt % of phospholipids which can bemade by the above method. Such a composition may contain at least 75 wt% of phospholipids, and even at least 90 wt % phospholipids. Such acomposition may, as it is described above, be used in the enrichmentcomposition of the invention. However, other uses of such a compositionis contemplated, for example as a dietary component in food products,including infant formulas, and nutritional compositions administeredparenterally or via tubes, or as a pharmaceutically active agent and asa component in cosmetics.

The marine lipid composition according to the invention can be providedin any suitable form including the forms described above for theenrichment composition.

EXAMPLE 1 Laboratory Scale Isolation of a Phospholipid-rich LipidComponent from Capelin Fish Meal

To 100 g capelin fishmeal was added 400 mL ethanol (about 99% v/v) andthe mixture was stirred at room temperature for 3 h. The meal residuewas separated by filtration and the ethanolic filtrate distilled invacuum on a rotary evaporator to obtain 10–11 g of a crude lipidfraction containing about 80 wt % lipid of the following composition: 54wt % phospholipids (PL), 4 wt % free fatty acids (FFA), and 42 wt %triglycerides (TG). The extraction procedure was repeated on theseparated meal residue which yielded a further 1.5 g of crude lipid.

The combined crude lipid fractions were purified by addition of ethanolat a wt.:vol. ratio of 1:5 and the resulting suspension was left tostand at 4° C. overnight causing a substantial part of the triglyceridesto precipitate. The ethanolic phase was separated and subjected todistillation using a rotary evaporator to yield a phospholipid enrichedfraction as a red-yellowish dense, syrupy material (6.5–6.8 g;containing about 71% lipids of the composition shown below. (Numbers inleft-most column refer to the number of carbons and double bond in thefatty acids of the lipid components, DHA is 22:6 and EPA 20:5):

PL TG FFA Total 71% 23% 6% 100% 14:0 4.1 12.1 5.7 4.3 16:0 22.5 14.530.3 20.8 16:1 4.9 16.7 6.2 6.4 18:0 0.0 0.0 3.7 1.3 18:1 9.6 16.9 15.513.4 18:2 0.0 2.4 0.0 1.4 18:3 0.0 0.0 0.0 0.6 18:4 2.3 7.5 0.0 2.6 20:12.3 6.2 5.2 4.2 20:4 0.0 0.0 0.0 0.6 20:5 23.5 11.4 15.6 13.2 22:1 0.03.8 0.0 3.5 22:4 0.0 0.0 0.0 22:5 0.0 0.0 0.0 1.0 22:6 30.8 6.9 17.919.4 100.0 100.0 98.5 92.7

EXAMPLE 2 Laboratory Scale Isolation of a Phospholipid-rich LipidComponent from Herring Fish Meal

Essentially the same procedure as described in Example 1 was used toobtain a phospholipid-rich component from herring meal with thefollowing lipid composition:

PL TG FFA Total 81% 5% 14% 100% 14:0 3.2 21.0 11.3 5.2 16:0 25.2 14.824.1 24.5 16:1 1.7 13.0 6.0 2.9 18:0 1.2 0.0 2.5 1.3 18:1 5.9 10.8 10.36.8 18:2 0.7 0.0 1.4 0.8 18:3 0.4 0.0 1.4 0.5 18:4 0.8 8.5 5.0 1.8 20:10.8 3.6 3.7 1.3 20:4 0.7 0.0 1.3 0.7 20:5 14.9 13.8 12.1 14.4 22:6 38.27.8 15.4 33.5 93.7 93.3 94.5 93.7

As seen above, herring meal provides a highly preferred material for aphospholipid-rich component according to the invention, with a highproportion of DHA-phospholipid.

The ethanolic extraction procedure of Examples 1 and 2 can be scaled upwith conventional methods by the person skilled in the art, similar towhat is described in Example 3 with a different solvent system.

EXAMPLE 3 Large Scale Isolation of a Phospholipid-rich Lipid Componentfrom Squid Mantles

Minced squid (150 kg) was added to 300 L of isopropanol and the mixturewas agitated rather vigorously for 4–6 h and left to stand overnight.Subsequently, the mixture was filtered and 300 L of hexane were added tothe filtrate and mixed. This resulted in two phases which were allowedto separate. The upper phase, which largely consisted of hexane andisopropanol was separated and subjected to distillation in severalrounds in vacuum using a 50 L rotary evaporator to yield a total of 2.2kg of a phospholipid enriched fraction as a brown-yellowish wax having aphospholipid content of about 65 wt % and the following total fatty acidcomposition:

14:0 1.9 16:0 28.3 16:1 0.6 18:0 2.9 18:1 3.2 18:2 0.2 18:3 0.0 18:4 0.220:1 2.7 20:4 1.4 20:5 13.8 21:5 0.0 22:1 0.0 22:6 40.4 95.5

EXAMPLE 4 Preparation of an Enrichment Composition for Fish Larvae PreyOrganisms

A composition for prey organisms such as Artemia species was prepared bycombining and mixing the following ingredients:

TABLE 4.1 phospholipid-rich component from squid mantles (Example 3) 9.7 g TG 4010 (TM), Croda, essentially triglycerides  78.0 g w/≈40 wt %DHA vitamin C (ascorbyl palmitate)  8.5 g co-emulsifer, BASF ChremophoreA25 (TM)  1.6 g Glucan Macroguard (TM) (immunostimulant)  0.8 g vitaminA (vitamin A palmitate, 1 mill i.u./g) 0.190 g vitamin E (DL-alphatocopherol acetate) 0.155 g vitamin B (thiamine hydrochloride)  1.2 gTBHQ (antioxidant) 0.036 g Ethoxyquin (antioxidant) 0.036 g Total   100g

The TG 4010 material used as a DHA-rich component in the composition isderived from fish oil-based material which is enriched for DHA, itcomprises 40 wt % DHA, about 10 wt % EPA and about 10 wt % other n-3HUFAs. The fatty acids are mostly in the form of triglycerides and thematerial has a very low free fatty acid content.

Other materials have been tested as sources of a DHA-rich component,such as TG 5010 (also from Croda) which has a DHA content of about 50 wt%, and enzymatically highly DHA-enriched triglycerides.

EXAMPLE 5 Use of Enrichment Composition for Cultivating Artemia

Artemia cysts were hatched under optimal conditions (in seawater, 27–29°C., pH about 8, oxygen content above 4 mg/L). The newly hatched naupliarArtemia were rinsed and put in 250 L tanks to give a density of200.000/L. Temperature was kept at 25–28° C., oxygen content at 5–6 mg/Land pH buffered at 7.5 with sodium bicarbonate (2 g/L). The tanks wereaerated by passing atmospheric air through perforated hoses at bottom oftanks. Enrichment composition as described in Example 4 was added to thetanks to a concentration of 0.2 g/L and the same amount added 10 hlater. 24 h after the first addition of enrichment composition theArtemia has the following lipid composition (31% dw (dry weight) oflipids):

PL TG FFA Total 16% 76% 8% 100% 14:0 8.8 1.0 3.1 0.8 16:0 15.0 8.8 36.011.1 16:1 2.6 3.2 3.1 2.5 18:0 6.4 2.7 6.3 4.2 18:1 25.2 15.6 13.0 17.118:2 4.2 3.5 1.8 3.3 18:3 13.2 19.2 6.5 14.7 18:4 2.2 3.1 1.7 2.4 20:11.6 1.0 0.0 0.9 20:4 2.8 2.1 0.0 2.2 20:5 12.5 10.2 4.4 9.5 22:1 0.0 0.00.0 22:4 0.0 1.1 0.0 1.2 22:5 0.0 1.0 0.0 1.1 22:6 4.6 20.0 14.8 18.999.0 92.5 90.7 90.0

The Artemia thus obtained has a highly enriched total concentration ofDHA in accordance with the invention and is thus particularly suitablefor feeding fish larvae such as halibut larvae.

EXAMPLE 6 Use of Enrichment Composition for Cultivating Artemia

Newly hatched Artemia were placed in 250 L tanks and same conditions asdescribed in Example 5. The Artemia were fed a lipid composition mixed 2wt % Chremophore A25 emulsifier. The lipid composition contained 50 wt %phospholipid composition of Example 3; 25 wt % ‘DHA-80’, essentiallytriglycerides comprising 80 wt % DHA, synthesized enzymatically fromglycerol and DHA fatty acid using lipase from Candida Antarctica (asdescribed in U.S. Pat. No. 5,604,119); and 25 wt % Lysi-22™ (Lysi hf,Iceland), a fish oil with 22 wt % DHA. The feed composition was added tothe tanks to a concentration of 0.2 g/L and the same amount added 12 hlater. 24 h after the first addition of enrichment composition theArtemia has the following lipid composition (34% dw lipids):

PL TG FFA Total 25% 72% 3% 100% 14:0 0.9 1.1 0.0 1.3 16:0 13.6 10.6 32.011.2 16:1 3.3 3.5 3.3 3.4 18:0 5.8 2.2 10.6 3.3 18:1 26.2 15.4 15.7 15.118:2 3.7 2.7 0.0 2.5 18:3 13.8 15.0 4.4 13.7 18:4 2.7 2.1 0.0 2.2 20:11.0 1.9 5.3 2.0 20:4 2.1 1.7 0.0 1.9 20:5 13.1 8.7 5.0 9.7 22:6 8.4 28.823.6 28.0 94.6 93.7 100.0 94.1

The Artemia obtained has a very highly enriched total concentration ofDHA (9.5 wt %) in accordance with the invention as well as otherfish-characteristic n-3 HUFAs, and is thus particularly suitable forfeeding fish larvae such as halibut larvae.

EXAMPLE 7 Use of Enrichment Composition for Cultivating Rotifers(Brachionus plichatilis)

Rotifers were reared under similar conditions as described in Example 5,they were fed with Isochrysis plankton and yeast and enriched for 6 h at27 C with an enrichment composition as described in Example 4, exceptthat triglycerides TG 5010™ from Croda were used instead of TG 4010, TG5010 containing about 50 wt % of DHA. The rotifers had the followinglipid composition (22% dw lipids):

PL TG FFA Total 32% 56% 13% 100% 14:0 6.6 7.8 3.3 6.9 16:0 25.9 4.9 15.213.0 16:1 1.9 2.5 1.3 2.2 18:0 3.6 5.7 2.7 4.7 18:1 4.5 4.5 5.5 4.7 18:24.9 0.3 2.0 2.0 18:3 3.1 3.2 1.9 3.0 18:4 2.2 6.2 2.2 4.4 20:1 1.2 1.91.5 1.6 20:4 5.0 2.3 2.2 3.2 20:5 10.1 14.7 14.8 13.4 22:6 25.6 38.840.4 35.2 94.7 92.7 93.0 94.3

The rotifers obtained have a very high total concentration of DHA, aswell as containing other n-3 HUFAs and has a very high phospholipidcontent, and thus exemplifies the efficacy of the composition accordingto the invention.

EXAMPLE 8 Comparison of Enrichment Compositions for Cultivating Artemia

Artemia cysts were hatched as in Example 5 and transferred tocultivation tanks where conditions were kept as in Example 3 (except forsome difference in temperature, see table). Enrichment compositions wereprepared similar as described in Example 4, i.e. with same additivesadded as in Table 4.1 such as emulsifier, vitamins and about 10%phospholipid-rich component from squid mantles as described in Example3. The bulk ingredient (about 80%) of the preparations were commerciallipid compositions as listed in Table 8.1. These are AlgaMac 2000™, DHASelco™, DC DHA™ and feed grade Cod Liver Oil (from Lysi, Iceland). Thepreparations were added to the tanks to a concentration of 0.2 g/L andthe same amount added 10 h later. 24 h after the first addition ofenrichment composition the Artemia has the following lipid composition:

TABLE 8.1 AlgaMac DHA composition 2000 Selco DC DHA Cod liver oil fromEx. 2 T during growth 20° C. 27° C. 27° C. 20° C. 27° C. % dw lipids 17%24% 22% 23% 31% 14:0 2.3 3.4 1.1 3.2 0.8 16:0 12.6 13.5 10.9 16.0 11.116:1 4.4 4.6 3.7 6.2 2.5 18:0 4.7 5.5 4.6 4.5 4.2 18:1 19.2 24.8 34.525.8 17.1 18:2 3.6 5.6 6.9 4.4 3.3 18:3 23.1 28.4 17.9 21.1 14.7 18:43.9 4.8 2.8 4.3 2.4 20:1 0.4 0.9 2.8 0.9 20:4 1.3 1.3 2.2 20:5 4.4 5.36.3 6.0 9.5 22:1 22:4 2.8 0.6 1.2 22:5 0.6 1.1 22:6 7.8 4.1 8.3 2.9 18.990.3 100.0 89.3 97.3 90.0

The Artemia enriched with the preferred composition according to theinvention has clearly a higher enriched total concentration of DHA inaccordance with the invention and is thus particularly suitable forfeeding fish larvae such as halibut larvae.

EXAMPLE 9 Use of HUFA- and Phospholipid-enriched Artemia forAquacultural Rearing of Halibut

Halibut larvae were first fed at 230–250° d. (‘°d’: multiplicationfactor of temperature (° C.) and days since hatching, e.g., at 5.2° C.250° d corresponds to 48 days.) Circular rearing tanks were used, either3.5 or 7 m³. Larvae were gradually acclimatized to a rearing temperatureof 11° C. and a light intensity of 300–500 lux. The larvae were fedArtemia twice per day, in the morning and in the late afternoon. TheArtemia was enriched with an enrichment composition according to theinvention 24 h before the morning feed, then stored at 13–15° C. foranother 7–8 h for the afternoon feed. Feed rations were adjusted toallow for good digestion of the Artemia. Microalgae (Isocrysis sp.) wereadded to the rearing water to reduce stress and facilitate maximumingestion rates. Slight aeration was applied in the center of the tanksto homogenize the water quality and the feed particles. Slight circularcurrent was acquired with the inflow to distribute the larvae. Waterexchange was increased from 1.2 times per 24 h in the beginning up to3.3 times per 24 h in the end. Larval rearing tanks were cleaned daily.

Survival rates of over 80% in one tank from start of feed to end oflarval stage were observed (90% excluding “gapers”: larvae with jawdeformity), and frequently survival rates between 65 and 75% have beenobserved. On average about 80% of juveniles showed correct pigmentation,but upto 96% correct pigmentation in one tank were observed. Correctpigmentation is defined as a normal pigmentation color on the ocularside and no pigmentation on the blind side. About 65% of juveniles onaverage but up to 80% in one tank showed correct eye migration, that ishaving both eyes on the ocular side. Ongoing experiments indicate thateven higher average survival and pigmentation rates are obtainable.

The results show that DHA-enriched prey organisms according to theinvention are particularly suitable for the rearing of aquatic speciessuch as halibut in terms of high survival rates and quality.

1. A composition for feeding prey organisms for use in aquaculture,comprising 2–75 wt % of a marine organism lipid component comprising atleast 25 wt % of phospholipids, and comprising about 5-about 99% of afurther lipid component comprising tri- and-or diacylglycerides, and atleast one additional component selected from the group consisting of anemulsifier, an immunostimulant, a vitamin, an antioxidant, a mineral,and arachidonic acid; the composition having a fatty acid compositioncharacteristic of fish such that the composition provides a DHA contentof at least 30 wt %, an EPA content in the range of 2–15%, and a contentof n-3 HUFA, other than DHA and EPA, which include 18:3, 18:4, 20:4, and22:5 fatty acids in the range of 2–25 wt %, the composition having aratio of DHA to EPA which is at least 3 and a free fatty acid content ofless than 10 wt %.
 2. A composition according to claim 1 where thephospholipid-containing component comprises at least 50 wt % ofphospholipids.
 3. A composition according to claim 2 where thephospholipid-containing component comprises at least 70 wt % ofphospholipids.
 4. A composition according to claim 1, wherein thefurther lipid component comprises a DHA content of at least 20 wt %. 5.A composition according to claim 4 where the further lipid componentcomprises a content of DHA of at least 30 wt %.
 6. A compositionaccording to claim 5 where the further lipid component comprises acontent of DHA of at least 50 wt %.
 7. A composition according to claim4 where the amount of the further lipid component is about 50-about 95wt %, calculated on the composition.
 8. A composition according to claim4 where the amount of the phospholipid-rich component is about 5-about50 wt %.
 9. A composition according to claim 4, wherein the furtherlipid component has a content of DHA of at least 40 wt %.
 10. Acomposition according to claim 1 comprising a DHA content of at least 40wt %.
 11. A composition according to claim 10 where the total content ofDHA is at least 50 wt %.
 12. A composition according to claim 11 whereinthe marine organism lipid component is obtained from a material selectedfrom the group consisting of fish eggs, squid mantles and a planktonicbiomass.
 13. A composition according to claim 10, wherein the totalcontent of DHA is at least 60 wt %.
 14. A composition according to claim1 wherein the marine organism lipid component is obtained from afishmeal.
 15. A composition according to claim 14 wherein the fishmealis selected from the group consisting of capelin meal, herring meal,menhaden meal, mackerel meal, anchovy meal, sardine meal, horse mackerelmeal, and blue whiting meal.
 16. A composition according to claim 14wherein the fishmeal is a high-quality commercial fish meal.
 17. Acomposition according to claim 1 which is selected from the groupconsisting of a powder, a granulate, a paste, and flakes.
 18. Acomposition according to claim 1 wherein the total content of HUFAs isat least 40 wt %.
 19. A composition according to claim 1 wherein thetotal content of n-3 HUFAs is at least 50 wt %.
 20. A compositionaccording to claim 1, wherein the amount of free fatty acids is lessthen about 5 wt %.
 21. A composition according to claim 1 that furthercomprises a pharmaceutically active substance.
 22. A compositionaccording to claim 21 wherein the pharmaceutically active substance isselected from the group consisting of an antimicrobial agent, animmunologically active substance and a combination hereof.
 23. Acomposition according to claim 22 wherein the immunologically activesubstance includes a vaccine.
 24. A composition according to claim 1,wherein the total content of HUFAs is at least 50 wt %.
 25. An emulsioncomprising as the lipid phase a composition according to claim
 1. 26. Anemulsion according to claim 25 comprising at least 50 wt % of thecomposition.
 27. A method of manufacturing a composition according toclaim 1 comprising the steps of separating from a marine organismmaterial a crude lipid component comprising triglycerides andphospholipids, followed by a phospholipid enrichment step comprisingadding, at a temperature where triglycerides do not precipitate, asolvent to the crude lipid component and cooling the mixture toprecipitate triglycerides and removing the precipitate to obtain a lipidfraction comprising at least 25 wt % of phospholipids as said marineorganism lipid component in said composition.
 28. A method according toclaim 27 wherein the marine organism material is selected from the groupconsisting of a fish meal, fish eggs, squid mantles, and planktonicbiomass.
 29. A method according to claim 28 wherein the fish meal isselected from the group consisting of capelin meal, herring meal,menhaden meal, mackerel meal, anchovy meal, sardine meal, horse mackerelmeal, and blue whiting meal.
 30. A method according to claim 27comprising mixing the lipid fraction comprising at least 25 wt %phospholipids with a further lipid component having a DHA content of atleast 20 wt %.
 31. A method according to claim 30 wherein the furtherlipid component is obtained by a process comprising contacting glyceroland a HUFA in the presence of a catalyst or an enzyme capable ofcombining the glycerol and HUFA, under conditions permitting a glycerideor a mixture of glycerides to be formed.
 32. A method according to claim31 where the enzyme is a lipase, including a lipase produced by Candidaantarctica.
 33. A method according to claim 27 wherein the lipidfraction has a phospholipid content of at least 50 wt %.
 34. A methodaccording to claim 27 wherein the lipid fraction has a phospholipidcontent of at least 75 wt %.